I propose to continue our ongoing studies on the regulation of neuronal ion channels. Modulation of ion channel properties is of fundamental importance for the long-term regulation of neuronal electrical activity. Our approach remains a reductionist one, and involves the use of combined biochemical, molecular, biophysical and physiological techniques to study channel modulation. We will focus during the next funding period on the modulation of two classes of ion-dependent potassium channels from rat brain: calcium-dependent potassium (KCa) channels and sodium-dependent potassium (KNa) channels. (1)We have studied modulation of brain KCa channels extensively in membrane patches and phospholipid bilayers, and have obtained evidence for endogenous protein kinase and phosphatase activities that are closely associated with some of these channels and that modulate their activity. We will extend these studies to characterize the endogenous kinase and phosphatase activities in detail, with the goal of understanding how they interact to regulate KCa channel activity. This will involve the use of pharmacological probes, in conjunction with single channel measurements. (2)We have demonstrated that KNa channels are widespread in mammalian brain and are also subject to modulation. Their activity runs down rapidly in detached membrane patches but not in cell-attached patches, indicating that some cytoplasmic factor(s) is necessary for the maintenance of KNa channel activity. We will study the mechanisms of KNa channel modulation, and compare them with mechanisms involved in KCa channel modulation. (3)We will exploit our recent surprising finding that a peptide corresponding to the amino-terminal region of Shaker-like potassium channels can interact with and block brain KCa and KNa channels as well. We will carry out a detailed biophysical study of these interactions, with the goal of identifying structural features that may be common between the Shaker-like and the ion-dependent potassium channels. I am excited by this unique opportunity to gain new insights into channel regulatory mechanisms by comparing the properties and modulation of two distinct classes of ion- dependent potassium channels.